The 220 kDa filamentous haemagglutinin (FHA) is a major adhesin of Bordetella pertussis and is produced from a large precursor designated FhaB. Although partly surface associated, it is also very efficiently secreted into the extracellular milieu. Its secretion depends on the outer membrane accessory protein FhaC. An 80 kDa N-terminal derivative of FHA, named Fha44, can also be very efficiently secreted in a FhaC-dependent manner, indicating that all necessary secretion signals are localized in the N-terminal region of FhaB. A comparison of predicted and apparent sizes of FHA derivatives, in addition to immunoblot analyses of cell-associated and secreted FHA polypeptides, indicated that FhaB undergoes N-terminal maturation by the cleavage of an 8-9 kDa segment. However, phenotypic analyses of translational lacZ and phoA fusions showed that this segment does not function as a typical signal peptide. Co-expression of the Fha44-encoding gene with fhaC also did not allow for secretion of Fha44 in Escherichia coli. High levels of secretion could, however, be observed when the OmpA signal peptide was fused to the N-terminal end of Fha44. Regardless of the OmpA signal peptide-Fha44 fusion point, the E. coli-secreted Fha44 had the same M(r) as that secreted by B. pertussis, indicating that the N-terminal proteolytic maturation does not require a B. pertussis-specific factor. Similar to FHA, the B. pertussis-secreted Fha44 contains an as yet uncharacterized modification at its N-terminus. This modification did not occur in E. coli and is therefore not required for secretion. The N-terminus of Fha44 secreted by E. coli was determined and found to correspond to the 72nd residue after the first in-frame methionine of FhaB. The N-terminal modification was also found not to be required for haemagglutination or interaction with sulphated glycoconjugates.

The gram-negative bacterium Bordetella pertussis has adapted specific secretion machineries for each of its major secretory proteins. In particular, the highly efficient secretion of filamentous hemagglutinin (FHA) is mediated by the accessory protein FhaC. FhaC belongs to a family of outer membrane proteins which are involved in the secretion of large adhesins or in the activation and secretion of Ca 2؉ -independent hemolysins by several gram-negative bacteria. FHA shares with these hemolysins a 115-residue-long amino-proximal region essential for its secretion. To compare the secretory pathways of these hemolysins and FHA, we attempted functional transcomplementation between FhaC and the Proteus mirabilis hemolysin accessory protein HpmB. HpmB could not promote the secretion of FHA derivatives. Likewise, FhaC proved to be unable to mediate secretion and activation of HpmA, the cognate secretory partner of HpmB. In contrast, ShlB, the accessory protein of the closely related Serratia marcescens hemolysin, was able to activate and secrete HpmA. Two invariant asparagine residues lying in the region of homology shared by secretory proteins and shown to be essential for the secretion and activation of the hemolysins were replaced in FHA by site-directed mutagenesis. Replacements of these residues indicated that both are involved in, but only the first one is crucial to, FHA secretion. This slight discrepancy together with the lack of functional complementation demonstrates major differences between the hemolysins and FHA secretion machineries.